Laurence Venisse

Platelet protease nexin-1, a serpin that strongly influences fibrinolysis and thrombolysis.

Background— Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the &agr;-granules of platelets. Methods and Results— In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1–deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1–deficient mice. Surprisingly, general physical health, after tissue plasminogen activator–induced thrombolysis, was much better in PN-1–deficient than in wild-type mice. Conclusions— Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator–mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

Macrophages and Platelets Are the Major Source of Protease Nexin-1 in Human Atherosclerotic Plaque

Objective—Protease nexin-1 (PN-1), a serpin constitutively expressed by vascular smooth muscle cells and endothelial cells, inhibits thrombin, plasminogen activators, and plasmin and can thus be expected to play a role in vascular biology. The present study addressed the question of PN-1 expression in human atherothrombosis. Methods and Results—Immunohistochemistry and biochemical studies confirmed that PN-1 was expressed at a moderate level in the medial layer of normal human arteries and showed that PN-1 expression was increased in atherothrombotic lesions. In early noncomplicated plaques, PN-1 was associated with infiltrating mononuclear cells. A strong PN-1 signal was observed in advanced lesions, principally in intraplaque hemorrhage-related structures. Monocytes/macrophages and platelets were identified as the main sources of PN-1 within atherothrombotic material. Isolated human monocytes and platelets both expressed high levels of active PN-1, and monocyte PN-1 expression was upregulated, at both messenger and protein levels, in response to stimulation by lipopolysaccharides. In contrast, PN-1 expression was downregulated during their differentiation into macrophages which were shown to produce degraded forms of PN-1. Conclusions—Platelets and monocytes/macrophages are a major source of PN-1 in human atherothrombotic plaques. PN-1 could thus represent a new actor in the evolution of atherosclerotic lesions.

Protease Nexin-1 Interacts With Thrombomodulin and Modulates Its Anticoagulant Effect

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1–overexpressing human aortic endothelial cells and PN-1–underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.

The serpin protease nexin-1 regulates vascular smooth muscle cell adhesion, spreading, migration and response to thrombin

Summary.  Background: Protease nexin‐1 (PN‐1) is an important physiological regulator of thrombin in the brain. PN‐1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. Objectives: In this study, we investigated the effect of PN‐1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. Results: Two clones exhibiting a two‐ to threefold increase in PN‐1 expression were selected and compared with untransfected and mock‐transfected cells. Overexpression of PN‐1 was observed to inhibit thrombin‐induced cell responses as indicated by a twofold decrease in induction of PAI‐1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN‐1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN‐1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. Conclusions: Our studies thus reveal that PN‐1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin‐induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.

Increased expression of protease nexin-1 in fibroblasts during idiopathic pulmonary fibrosis regulates thrombin activity and fibronectin expression

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-β, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.

Endothelial Protease Nexin-1 Is a Novel Regulator of A Disintegrin and Metalloproteinase 17 Maturation and Endothelial Protein C Receptor Shedding via Furin Inhibition

Objective—Human protein C is a plasma serine protease that plays a key role in hemostasis, and activated protein C (aPC) is known to elicit protective responses in vascular endothelial cells. This cytoprotective activity requires the interaction of the protease with its cell membrane receptor, endothelial protein C receptor. However, the mechanisms regulating the beneficial cellular effects of aPC are not well known. We aimed to determine whether a serine protease inhibitor called protease nexin-1 (PN-1) or serpinE2, expressed by vascular cells, can modulate the effect of aPC on endothelial cells. Approach and Results—We found that vascular barrier protective and antiapoptotic activities of aPC were reduced both in endothelial cells underexpressing PN-1 and in endothelial cells whose PN-1 function was blocked by a neutralizing antibody. Our in vitro data were further confirmed in vivo. Indeed, we found that vascular endothelial growth factor–mediated hyperpermeability in the skin of mice was markedly reduced by local intradermal injection of aPC in wild-type mice but not in PN-1–deficient mice. Furthermore, we demonstrated a previously unknown protective role of endothelial PN-1 on endothelial protein C receptor shedding. We provided evidence that PN-1 inhibits furin, a serine protease that activates a disintegrin and metalloproteinase 17 involved in the shedding of endothelial protein C receptor. We indeed evidenced a direct interaction between PN-1 and furin in endothelial cells. Conclusions—Our results thus demonstrate an original role of PN-1 as a furin convertase inhibitor, providing new insights for understanding the regulation of endothelial protein C receptor–dependent aPC endothelial protective effects.

Clearance of plasmin–PN-1 complexes by vascular smooth muscle cells in human aneurysm of the ascending aorta

Plasminogen is a circulating zymogen which enters the arterial wall by radial, transmural hydraulic conductance, where it is converted to plasmin by tissue plasminogen activator t-PA on an activation platform involving S100A4 on the vascular smooth muscle cell (vSMC) membrane. Plasmin is involved in the progression of human thoracic aneurysm of the ascending aorta (TAA). vSMCs protect the TAA wall from plasmin-induced proteolytic injury by expressing high levels of antiproteases. Protease nexin-1 (PN-1) is a tissue antiprotease belonging to the serpin superfamily, expressed in the vascular wall, and is able to form a covalent complex with plasmin. LDL receptor-related protein-1 (LRP-1) is a scavenger receptor implicated in protease-antiprotease complex internalization. In this study, we investigated whether PN-1 and LRP-1 are involved in the inhibition and clearance of plasminogen by the SMCs of human TAA. We demonstrated an overexpression of S100A4, PN-1, and LRP-1 in the medial layer of human TAA. Plasminogen activation taking place in the media of TAA was revealed by immunohistochemical staining and plasmin activity analyses. We showed by cell biology studies that plasmin-PN-1 complexes are internalized via LRP-1 in vSMCs from healthy and TAA media. Thus, two complementary mechanisms are involved in the protective role of PN-1 in human TAA: one involving plasmin inhibition and the other involving tissue clearance of plasmin-PN1 complexes via the scavenger receptor LRP-1.

Hematopoietic protease nexin-1 protects against lung injury by preventing thrombin signaling in mice

Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.

Selective neutralization of the serpin protease nexin-1 by a specific monoclonal antibody

Protease nexin-1 [PN-1; also termed serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2; SERPINE2] is a serpin expressed by many cell types (Bouton et al, 2012). PN-1 is known to inhibit a large spectrum of serine proteases, particularly proteases involved in coagulation and fibrinolysis, such as thrombin and proteases of the plasminergic system, respectively (Scott et al, 1985; Evans et al, 1991). PN-1 is stored in platelets and secreted as an active antiprotease during platelet activation (Boulaftali et al, 2010). Therefore, it plays a crucial role in the regulation of thrombus formation and lysis. Indeed, anticoagulant and antifibrinolytic properties of PN-1 have been demonstrated in vivo using mice models of vascular thrombosis and thrombolysis, respectively (Boulaftali et al, 2010, 2011). These data indicate that PN-1 could be an attractive target to prevent or limit thrombotic disorders. Very few inhibitors of PN-1 have been characterized. Kousted et al (2014) previously described monoclonal antibodies binding human PN-1 and abolishing all protease inhibitory activities of PN-1. However, the dual anticoagulant and antifibrinolytic role of PN-1 hampers the clinical use of a non-selective PN1 inhibitor. For this reason, it is important to determine if inhibitors targeting solely one specific anti-proteolytic activity of PN-1 can be developed. Here we describe the characteristics of a new monoclonal antibody raised toward human PN-1, MA-48H11, produced according to the procedure reported by Galfre and Milstein (1981). MA-48H11 is of the IgG2b kappa isotype and its reactivity with human PN-1 was determined with a standard one-sided enzyme-linked immunosorbent assay (ELISA), using recombinant human PN-1 antigen for capture and a horseradish peroxidase (HRP)-conjugated rabbit antimouse polyclonal antibody. The affinity constant for binding between MA-48H11 and human PN-1 was determined by surface plasmon resonance (SPR) analysis. The Ka value was 4 7–4 9 9 10 mol/l (Kd: 2 1–2 2 9 10 10 mol/l). The specificity of MA-48H11 to PN-1 was confirmed by the absence of binding to plasminogen activator inhibitor-1 (PAI-1) by ELISA and to antithrombin (AT) by SPR analysis. The ability of MA-48H11 to inactivate the inhibitory activity of PN-1 was tested in chromogenic assays for the two central proteases of coagulation and fibrinolysis, i.e. thrombin and plasmin, respectively. The presence of a 20fold molar excess of MA-48H11 over PN-1 did not prevent the inhibition of plasmin amidolytic activity by PN-1 (Fig 1A). In contrast, MA-48H11 significantly abolished the inhibition of thrombin amidolytic activity by PN-1 (Fig 1B), with 50% inhibitory concentration of 2 2 0 5 nmol/l. A

A034 Effet anticoagulant et antithrombotique de la PN-1 plaquettaire

La protease nexine 1 (PN-1), serpine tissulaire, est le plus puissant inhibiteur connu de la thrombine in vitro et inhibe l’uPA et le t-PA. Cependant son role physiologique dans l’hemostase et la thrombose n’est pas etabli. Notre objectif a ete de caracteriser la PN-1 plaquettaire quant a sa localisation et a sa fonction regulatrice de la thrombine et de l’uPA in vitro et in vivo. La PN-1 a ete analysee par cytometrie en flux et western blot. L’inhibition de la thrombine et l’uPA a ete mesuree par un test amidolytique. La generation de thrombine a ete analysee par thrombinographie sur plasma riche en plaquettes (PRP). La sensibilite des plaquettes sauvages (WT) et PN-1-/- a la thrombine a ete mesuree par FACS et agregation plaquettaire. Le role de la PN-1 plaquettaire dans la thrombose a ete d’abord etudie ex vivo en chambre de perfusion sur matrice de collagene et in vivo par microscopie intravitale. La PN-1 est detectee a la membrane et dans les granules α des plaquettes. La PN-1 secretee lors de l’activation plaquettaire inhibe la thrombine et l’uPA. La generation de thrombine en PRP en absence de PN-1 est significativement augmentee (anticorps bloquant ou plaquettes PN-1-/-) indiquant que la PN-1 plaquettaire inhibe la thrombine et sa generation. La mesure de l’expression de la Pselectine et le taux d’agregation des plaquettes PN-1-/- montre une hypersensibilite a la thrombine par rapport aux plaquettes WT. Ex vivo, la formation de thrombi sur une surface de collagene en condition de flux est egalement augmentee en absence de PN-1. In vivo, le deficit en PN-1 resulte en une acceleration de l’occlusion vasculaire dans un modele de thrombose experimentale. Nous montrons pour la premiere fois que la PN-1 plaquettaire, stockee dans le granule α a une activite anticoagulante via l’inhibition de la thrombine et de sa generation. La PN-1 plaquettaire inhibe egalement l’uPA aussi bien que le PAI-1 plaquettaire. Nous montrons egalement pour la premiere fois que la PN-1 plaquettaire a une activite antithrombotique et joue un role regulateur sous estime dans la thrombose.